A study of fast and metastable dissociations of adenine-thymine binary-base oligonucleotides by using positive-ion MALDI-TOF mass spectrometry

Abstract

In the present study, fast and metastable dissociations of a number of adenine-thymine binary-base oligonucleotides under the conditions of UV matrix-assisted laser desorption/ ionization mass spectrometry were investigated. 2-Aminobenzoic acid/ammonium fluoride (ABA/NH 4 F) matrix system was used. The spectra obtained under metastable and fast dissociation conditions exhibit distinctive dissociation products. From the post-source-decay analysis, all oligonucleotides underwent predominantly metastable dissociations at the 3' C-O linkages to form [a n -B] ϩ and w n ϩ complimentary ion series. Based on the present results, the so-called "[w n ϩ80] ϩ " ions were postulated to be the complimentary [z (8Ϫn) AH] ϩ ions rather than the expected phosphate rearrangement products. In addition, these oligonucleotides were found to generate fast dissociation products of b n ϩ , d n ϩ , w n S tructural characterization of short-chain nucleic acids has recently attracted much attention. They can serve as useful tools in molecular biology, as gene probes for diagnosis, or as potential drugs interacting at the level of nucleic acids. Typical short-chain DNA sequencing methods involve the use of the enzymatic or chemical cleavage algorithms [1][2][3] to produce DNA ladder products. The DNA fragments generated are then separated and identified by HPLC or gel electrophoresis. Structural verification of oligonucleotides using these methods is time-consuming and is sometimes impossible for modified oligonucleotides. Recent developments in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry [4 -9] have stimulated interest in replacing HPLC/gel electrophoresis by mass spectrometry in order to increase the speed and accuracy of the sequence determination. However, this approach still needs the time-consuming chemical or enzymatic reactions for producing sequence specific fragment ladder. A potentially more efficient method would be to obtain the DNA sequence information from fragmentation patterns in mass spectrometry through dissociation. In the present study, we attempt to evaluate the potential use of two rather direct mass spectrometric sequencing methods, namely fast and metastable dissociations for DNA sequence analysis.Fast fragmentation differs from metastable dissociation at the time of bond cleavage after the excitation of the precursor ions. In mass spectrometry, fragments are produced in a much shorter time as compared to the ion extraction time of the mass spectrometer. In the case of time-of-flight mass spectrometer, these fragment ions are therefore registered at their true masses in both linear and reflectron mode of detection. Metastable fragments, on the other hand, are usually formed at a much longer time scale (i.e., Ͼ500 ns) and are formed beyond the ion source region and before reaching the detector. Since the metastable ions are formed with the same velocity of the precursor ions, they are registered at the same mass-to-charge ratio of the precursor ion in a linear TOF instrum...

MALDI In-Source Decay, from Sequencing to Imaging

“…A base loss is considered to be a critical step of this "thermal" fragmentation pathway [75,76]. Alternatively, a pathway of electronic energy transfer was also proposed to occur in positive ion mode with 2-aminobenzoic acid and leads to the formation of supplementary ions (d-, b-, and y-ions) [77]. Concerning applications of ISD in the nucleic acids field, ISD was successfully applied by Wang et al to sequence covalently modified (phosphorothioate instead of phosphodiester bonds) oligonucleotides [78] but failed to allow locating covalent rhodium adducts on DNA [79].…”

Development of new methodologies for the mass spectrometry study of bioorganic macromolecules

“…The ability to handle large DNA and RNA fragments should facilitate high-throughput screening applications for genotyping and clinical diagnosis. It has also been shown that the PSD technique is effective to investigate oligonucleotide fragmentation processes (Chan, Fung, & Li, 2002).…”

Effective quenching of fragment formation in negative ion oligonucleotide matrix‐assisted laser desorption/ionization mass spectrometry through sodium adduct formation

“…There has been substantial effort to understand the fragmentation mechanism behind the prompt and metastable decay of oligonucleotides in MALDI 4–7, 9, 23–32. Much of this work was reviewed by Wan and Gross in 2001 and by Wu and McLuckey in 2004 6, 32.…”